1. Field of Invention
This invention is directed to methods for the detection and/or quantitation of the SARS virus, reagents and test kits containing the same for use in the method.
2. Background of the Invention
Severe acute respiratory syndrome (SARS) is one of the most recent emerging infectious diseases. The cause of SARS has been identified as a new coronavirus—a virus within the family Coronaviridae—designated as the “SARS coronavirus” (SARS-CoV) [1, 2] by the World Health Organization, following assessment of causation according to Koch's postulates, including monkey inoculation [3]. The coronaviruses are enveloped positive single-stranded RNA viruses with genomes approximately 30 kb in length—the largest of any of the RNA viruses—that replicate in the cytoplasm of host cells without going through DNA intermediates. Coronaviruses have been reported to cause common colds in humans, and to cause respiratory, enteric, and neurological diseases, as well as hepatitis, in animals. Human coronaviruses are usually difficult to culture in vitro, whereas most animal coronaviruses and SARS-CoV can easily be cultured in Vero E6 cells [4]. There are three groups of coronaviruses: Groups 1 and 2 encompass mammalian viruses, whereas Group 3 encompasses avian viruses. Within each group, the coronaviruses are classified into distinct species according to host range, antigenic relationships, and genomic organization. Human coronaviruses (HCoVs) were previously reported to belong in Group 1 (HCoV-229E) and Group 2 (HCoV-OC43), and are responsible for mild respiratory illnesses.
Recently, two independent groups, one at the British Columbia Cancer Agency (BCCA) in Canada [5] (Tor2 isolate), and the other at the Centers for Disease Control and Prevention (CDCP) in the United States [6] (Urbani isolate), were first to obtain full genomic sequences of SARS-CoV. Phylogenetic analyses, based on the genome sequences, revealed that both isolates were distantly related to previously characterized coronaviruses, including the two previously isolated nonpathogenic human coronaviruses strains, HCoV-C43 and HCoV-229E. The genome of the Tor2 CoV isolate is 29,751 nucleotides long, and the genome of the Urbani CoV isolate is 29,727 nucleotides long, and their sequences differ at only 24 nucleotide positions. The genomic organization of both isolates is characteristic of coronaviruses having the following typical gene order: 5′-replicase (rep), spike (S), envelope (E), membrane (M), and nucleocapsid (N). The SARS-CoV rep gene, which is approximately 20,000 nucleotides long, is predicted to encode two polyproteins (ORF1a and ORF1b) that undergo proteolytic processing, resulting in several nonstructural proteins. There are four genes downstream of rep that encode the structural proteins S, E, M, and N.
The genome of SARS-CoV has several distinct genomic characteristics that distinguish it from other coronavirus isolates and that could be of biological significance. The gene encoding hemagglutinin-esterase, which is present between ORF1a and S in Group 2 coronaviruses (and in some Group 3 coronaviruses) is absent, and so is the short anchor of the S protein. Furthermore, the short anchor of the S protein, the specific number and location of the small ORFs, and the presence of only one copy of PLPPRO provide a combination of genetic features that readily distinguish SARS-CoV isolates from previously the described coronaviruses [5, 6]. There are several publications that describe reverse-transcriptase polymerase chain reaction assays (RT-PCR assays) for the detection of SARS-CoV.
Perris et al. [2] developed as RT-PCR assay that identifies the virus from nasopharyngeal aspiration samples obtained from patients infected with SARS-CoA. Total RNA from clinical samples is reverse transcribed in the presence of random hexamers, and the resulting cDNA is amplified with primers 5′-TACACACCTCAGCGTTG-3′ (SEQ ID NO: 86) and 5′-CACGAACGTGACGAAT-3′ (SEQ ID NO: 87). To determine the genetic sequence of an unknown RNA virus, they perform a random RT-PCR assay. Total RNA from virus-infected fetal rhesus kidney cells were isolated, reverse transcribed with primer 5′-GCCGGAGCTCTGCAGAATTCNNNNNN-3′ (SEQ ID NO: 88), and the resulting cDNA was amplified with primer 5′-GCCGGAGCTCTGCAGAATTC-3′ (SEQ ID NO: 89).
Ksiazek et al. [1] developed a reverse transcription and real-time PCR assay to identify SARS-CoV. Oligonucleotide primers used for amplification and sequencing of the SARS-related coronavirus were designed from alignments in open reading frame 1b of the coronavirus polymerase gene sequences. They used the primer pair IN-2 (+) 5′-GGGTTGGGACTATCCTAAGTGTGA-3′ (SEQ ID NO: 90) and IN-4 (−) 5′-TAACACACAACICCATCATCA-3′ (SEQ ID NO: 91), which was previously designed to hybridize to conserved regions of the open reading frame 1b (ORF1b), in order to achieve broad reactivity with coronavirus/genus. These primers were used to amplify DNA from SARS isolates, and the amplicon sequences obtained were used to design SARS-specific primers Cor-p-F2 (+) 5′-CTAACATGCRRAGGATAATGG-3′ (SEQ ID NO: 92), Cor-p-F3 (+) 5′GCCTCTCTTGTTCTTGCTCGC-3′ (SEQ ID NO: 94), which were used in turn to test patient specimens. Drosten et al. [4] used a PCR-based random-amplification procedure to genetically characterized a 300-nucleotide-long SARS-CoV genomic segment. On the basis of the sequence that was obtained, conventional real-time PCR assays for specific detection SARS-CoV ORF1b were established. Poon et al. [7] developed an RT-real-time-PCR assay. Total RNA isolated from stool specimens from SARS-CoV-infected individuals is reverse transcribed with random hexamers and resulting can is amplified with primers coro3 5′-TACACACCTCAGCGTTG-3′ (SEQ ID NO: 86) and CORO4 5′-CACGAACGTGACGAAT-3′ (SEQ ID NO: 87), which recognize a region of the viral polymerase gene. It is important to note that these authors acknowledges in their publication that the primers that they use in their assay can cross-react with the nonpathogenic human coronavirus strain HCoV-OC43.
SARS-specific PCR priers and diagnostic procedures were developed in several World Health Organization laboratories for the amplification of a region of the open reading frame 1b of the SARS-CoV polymerase gene sequence. These primers are currently being assessed to determine their relative performance and sensitivity with difference specimens obtained at different times over the course of illness. Lipkin and Breise have announced they develop a PCR-based SARS diagnostic that detects a SARS-CoV gene that is present in multiple copies, but no further information is available in the literature.
Problems with the prior art that the current invention is designed to solve. The main problems with current molecular diagnostic assays are: a) failure to consider the intrinsically polymorphic nature of coronaviruses, including the current SARS-CoV strains originated from the Tor2 and Urbani isolates—the ability of the virus to mutate and recombine during the period of time it is within the infected individual, and during horizontal transmission; and b) failure to account for the possibility of continuous and/or multiple introduction of non non-genetically identical SARS-CoV strains into the human population.
A characteristic of RNA viruses is their high rate of genetic mutation, which leads to evolution of new viral strains, and is well-established mechanism by which viruses escape the immune system. Coronaviruses, including SARS-CoA, are quite sloppy when it comes to replicating their genetic material, producing one error for every 10,000 nucleotides that they copy, which is roughly the same error rate as occurs during the replication of human immunodeficiency virus, HIV-1. Coronavirus RNA polymerase sometimes jumps between multiple copies of the viral genome that are present in an infected cell. Therefore, each new genome is actually copied from several templates, reducing the chance that any given mutation will become well established in the viral population. Moreover, if one of these jumps is imprecise, a whole chunk of genome can get skipped, resulting in the deletion of part of an important gene. The consequences can be dramatic, particularly if the change affects the protein spikes that enable the virus to bind to the surface of the host's cells. For example, in 1984 a new respiratory sickness appeared in European pig farms. It turned out to be a deletion mutant of a coronavirus that previously had infected piglets' stomachs 8. It possessed an altered spike protein that enabled the virus to infect a different cell type. Although the new disease was not generally lethal, it has since spread worldwide, and has complicated the diagnosis of the gut disease. Another example is the recent introduction of SARS-CoV into the human population. It is likely that a genetic deletion may have helped the SARS virus strains to make the transition from its animal reservoir to humans. Genetics analyses of the viral strains found in animals for sale in the Southern Chinese markets indicated that these SARS strains lacked 29 nucleotides in the gene encoding a protein of unknown function and the protein product of this gene is attached to the inside of the virus's coat protein. Furthermore, in a recent publication, full genome sequences of 14 isolates from SARS-CoV-infected patients in Singapore, Toronto, China and Hong Kong were compared, and 14 mutations were revealed 9. In one respect, this finding may be viewed as indicating that SARS virus fails to mutate; however, this virus has so far encountered little resistance from it new human hosts, and there has, therefore, been little selective pressure to cause new mutants to be retained. SARS-CoV will probably not remain as stable as it has been so far. Our immune systems could force changes, similar to the changes that frequently occur in flu viruses. In summary, we deem it prudent to develop a new SARS-CoV diagnostic assay that accounts for the genetically polymorphic nature of coronaviruses, including SARS-CoV.